DNA complexes with dyes designed for energy transfer as fluorescent markers

ABSTRACT

Heteromultimeric fluorophores are provided for binding to DNA, which allow for the detection of DNA in electrical separations and preparation of probes having high-fluorescent efficiencies and large Stokes shifts. In addition, by appropriate choice of fluorescent molecules, one can use a single narrow wavelength band excitation light source, while obtaining fluorescent emissions having sufficient separation to be readily discriminated.

CROSS-REFERENCE TO GOVERNMENT GRANT

This invention was made with Government support under Grant Contract No.DE-FG-91ER61125 awarded by the Department of Energy. The Government hascertain rights in this invention.

This application is a continuation of application Ser. No. 08/340,749,filed Jan. 23, 1995, U.S. Pat. No. 5,646,264, which is a continuation ofapplication Ser. No. 08/009,704, filed on Jan. 27, 1993, U.S. Pat. No.5,401,847 which is a continuation-in-part of application Ser. No.07/831,823, filed Feb. 6, 1992 abandoned which is a continuation-in-partof application Ser. No. 493,307, filed Mar. 14, 1990 abandoned.

INTRODUCTION

1. Technical Field

The field of this invention is fluorescent compositions.

2. Background

Detection of fluorescent signals finds wide application in a variety ofsituations and under a variety of conditions. Fluorescence has manyadvantages as a means of generating a detectable signal. Fluorescencedoes not suffer from the many disadvantages of a radio-active label,while in many cases it provides for a high level of sensitivity.Instrumentation for detection of fluorescence is readily available andfluorescent labels have found application in such diverse situations asimmunodiagnostics, detection of nucleic acid bands and protein bands ingel electrophoresis and in fluorescence activated cell sorters. Thesensitivity of the fluorescent signal depends upon a number of factors:the possibility of self-quenching; the effect of other moleculesassociated with the fluorescent molecule on the quantum efficiency ofthe fluorescence; the effect of the medium on the quantum efficiency andfluorescence characteristics of the fluorescer; the stability of thefluorescer to light; the ability to remove background fluorescence; andthe nature of the light source.

For many applications one wishes to have a number of distinguishablefluorescers, so that one can detect different characteristics of asystem. For example, in the FACS, there may be an interest inidentifying the presence of two characteristics of the cell or othercomposition. In the hybridization of DNA, one may wish to observe twodifferent DNA sequences, as observed in a gel, on a plate, or the like.Frequently, it is very difficult to obtain fluorescers having emissionmaxima which are sufficiently different so as to be differentiable whileallowing for excitation at the same wave length. There is, therefore,substantial interest in identifying fluorescent markers which permitmultiplex determinations by providing for readily differentiable,fluorescent emission maxima, while allowing for excitation with a narrowband radiation source.

Relevant Literature

The following references describe DNA intercalating fluorescent dimersand their physical characteristics: Gaugain et al., Biochemistry 17,5071-5078, 1978; Gaugain et al., Biochemistry 17, 5078-5088, 1978;Markovits et al., Anal. Biochemistry 94, 259-269, 1979; MarkovitsBiochemistry 22, 3231-3237, 1983; and Markovits et al., Nucl. Acids Res.13, 3773-3788, 1985. Interaction of various intercalating compounds withnucleic acids is reviewed by Berman and Young, Ann. Rev. Biophys.Bioeng. (1986) 10:87-224. Retention of ethidium bromide onelectrophoresis of the dye with DNA or RNA is described by Angemullerand Sayavedra-Soto, Biotechniques 8, 36, 1990 and Rosen andvilla-Komaroff, Focus 12, 23, 1990 (see also Glazer et al. (1990) Proc.Natl. Acad. Sci. USA 87:3851-3855; Rye et al. (1991) Nucl. Acids Res.19:327-333; Quesada et al. (1991) BioTechniques 10:616-625; Rye et al.(1992) Nucl. Acids Res. 20:2803-2812.

SUMMARY OF THE INVENTION

Novel fluorescent compositions and their use are provided, where thefluorescent compositions are characterized by strongly binding to doublestranded DNA having a multiplicity of positive charges, having at leasttwo fluorophoric moieties, where one moiety is capable of efficientlyquenching the fluorescence (excitation energy) of the moiety emitting ata lower wave length; the intercalated fluorescent composition providesfor emission efficiencies substantially in excess of the presentfluorophore; and ratios of observed fluorescence emission between thehigher wave length emitting fluorophore and the lower wave lengthemitting fluorophore are at least about 1.2 to 1. These compositionsfind use as fluorescent markers for DNA, in combination with DNA aslabels for other molecules, and in the analysis of various systems,where multiplex fluorescent analysis is desired.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a synthetic scheme for the synthesis of thiazole monomerintermediates;

FIG. 2 is a synthetic scheme for the synthesis of thiazole dimers (TOTOand TOTAB);

FIG. 3 is a synthetic scheme for the synthesis of thiazole-ethidiumdimer (TOED); and

FIG. 4 is a synthetic scheme for the synthesis of fluorescein-ethidiumheterodimer (FED).

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

Novel methods are provided employing heterodimeric fluorescentcompounds, by themselves or intercalated into double stranded nucleicacids, where the compositions are characterized by having two differentfluorophore moieties joined by a linking group comprising at least twopositive charges, advantageously used together or in conjunction with ahomodimer. The fluorophore moieties of the heterodimer are related inthat a first fluorophore moiety has an absorption maximum in solution atless than about 500 nm, usually less than about 450 nm, and generallygreater than about 275 nm, usually greater than about 300 nm. Theemission maximum for the second fluorophore moiety in solution willgenerally be greater than 350 nm, usually greater than about 400 nm, andless about 600 nm, usually less than about 550 nm. By contrast, theabsorption maximum when intercalated in DNA for the first fluorophoremoiety will usually be at least about 375 nm, usually at least about 400nm, while, more usually at least about 450 nm. The emission maximum forthe second fluorophore moiety when intercalated in DNA will generally beat least about 450 nm, more usually at least about 500 nm and usuallyless than about 750 nm, more usually less than about 700 nm. At leastone of the fluoropores will be capable of strongly binding to, usuallyintercalating, dsDNA.

The second fluorophore moiety will be further characterized, whenassociated with dsDNA, by having an absorption maximum at least 25 nmgreater than the first fluorophore moiety to which it is combined andusually not greater than about 100 nm, more usually not greater thanabout 75 nm. The second fluorophore will be capable of quenching atleast about 80% of the fluorescence which would otherwise be observedfrom the first fluorophore under the conditions of irradiation, when thesubject composition is intercalated into dsDNA. In order to haveefficient quenching, there will be overlap between the emission spectrumof the donor and the absorption spectrum of the recipient or secondfluorophore. Usually, the ratios of observed fluorescence emissionbetween the higher wave length emitting fluorophore and the lower wavelength emitting fluorophore will be at least 1.2, more usually at leastabout 2, and may be 5 to 1, or more. The Stokes shift between thewavelength of the irradiation, where a narrow wavelength band isemployed, particularly a laser, and the fluorescent emission maximumwill be at least about 75 nm, more usually at least about 100 nm,preferably greater than about 125 nm. It is advantageous to have a donorchromophore with an extinction coefficient at the excitation wavelengthto maximize the effectiveness of light absorption.

While donor fluorophores may have extinction coefficients as low asabout 2000, desirably the extinction coefficient will be at least 5000,preferably at least 10,000, and may be 50,000 or more.

The subject heterodimers have novel fluorescent properties. The emissionof the absorbing fluorophore is at least about 80%, usually at leastabout 85% quenched. In addition, when bound solely to primary bindingsites of dsDNA, the emission of the emitting fluorophore is at leastabout 2 times, usually at least about 7 times, brighter (as great) asthe emission maximum of the homodimer of the emitting fluorophores underthe same conditions, except for the wavelength of the excitation light.Thus, fluorescence yield can be greatly increased with a weaklyabsorbing fluorescer.

The subject compositions may comprise two or more fluorophore moieties,where a pair of the fluorophores are related in accordance with theranges indicated above. The other fluorophores may be the same ordifferent from the pair of fluorophores. If different, the fluorescentproperties will usually be governed by the ranges indicated for the pairof fluorophores. Where the fluorophores are different, each fluorophorewould have a differential absorption maximum in relation to the nextsuccessive fluorophore in accordance with the above-indicated ranges.That is, if there were three different fluorophore moieties, there wouldbe a difference in absorption maxima of at least 25 nm between the firstand second and at least 25 nm between the second and third, and so on.Usually, the molecules would not have more than about four differentfluorophores, more usually not more than about three differentfluorophores. It is not essential that there be any particular order inwhich the fluorophores are linked, so that they may be linked in alinear manner, from a central linking group having spokes radiating froma center, to a cyclic molecule or other convenient syntheticintermediate, so long as the distance between the fluorophores allowsfor energy transfer.

The linking group will be characterized by having at least two groups orfunctionalities that can carry a positive charge or are charged, forexample, amino groups or ammonium groups, sulfonium groups, etc., forthe most part comprising nitrogen or sulfur as the positive heteroatom.The linking chain may be of a length to allow for simultaneousintercalation to adjacent monomeric units dsDNA, in this case usuallyproviding a length of at least about 10 Angstroms, usually having atleast about 9 atoms, more usually at least about 10 atoms in the chain,and usually not more than about 26, usually not more than about 20atoms, between fluorescent units, counting the shortest chain where acyclic linking group is involved.

The linking group will usually be aliphatic or alicyclic, having from0-8, more usually from 0-6, preferably from about 2-6 heteroatoms in thechain, particularly heteroatoms that would provide for a positivecharge. There will normally be at least a total of two positive charges,more usually not more than eight positive charges, more usually not morethan about six positive charges on the linking group.

The fluorophoric moieties may be cyclic, polycyclic, particularlypolycyclic aromatic having at least two rings, and not more than aboutsix rings, more usually not more than about five rings, where at leasttwo of the rings are fused, usually not more than four of the ringsbeing fused. The aromatic compound may be carbocyclic or heterocyclic,particularly having from 1-3, more usually 1-2 nitrogen atoms asheteroannular atoms. Other heteroannular atoms include oxygen and sulfur(chalcogen).

The rings may be substituted by a wide variety of substituents, whichsubstituents may include alkyl groups of from 1-4 carbon atoms, usually1-2 carbon atoms, oxy, which includes hydroxy, alkoxy and carboxy,generally of from 1-4 carbon atoms, amino, including mono- anddisubstituted amino, particularly mono- and dialkyl amino, of from 0-8,usually 0-6 carbon atoms, thio, particularly alkylthio from 1-4, usually1-2 carbon atoms, cyano, non-oxo-carbonyl, such as carboxy andderivatives thereof, particularly carboxamide or carboxyalkyl, from 1-8,usually 1-6 carbon atoms, oxo-carbonyl or acyl, generally from 1-4carbon atoms, halo, particularly of atomic number 9-35, etc.

Fluorophore moieties of particular interest will involve two ringsystems, which are joined by a bond or a linking group having one ormore ethylenic groups which are in conjugation with the aromaticmoieties. Aromatic groups of interest include benzimidazole,benzthiazole, benzoxazole, quinoline and acridine. Illustrative groupsinclude thiazole orange, thiazole blue, ethidium, fluorescein, acridine,phenanthridine, xanthenes, and particularly fluorones.

Couples of fluorophore moieties include thiazole orange and thiazoleblue, thiazole orange and ethidium, fluorescein and ethidium, acridineand ethidium, etc. However, acridine has a very low extinctioncoefficient above 320 nm and is not a donor of choice. Acridine couldserve as a donor for thiazole orange or for fluorescein, but would givemarginal gains in fluorescence emission.

Compounds can be prepared from alkylene polyamines, where the alkylenegroups are from 2-10 usually 2-6 carbon atoms, and haloalkyl- orpseudohaloalkyl substituted fluorescent polycyclic aromatic compounds,to provide for ternary or quaternary amino groups. The amino groups maybe quaternized with any convenient alkylation agent, either before orafter reaction with the fluorescent compound or may be preparedinitially as ternary amines using alkyl amines, where the alkyl groupwill be of from about 1-6, usually 1-3 carbon atoms.

These compounds find use as labeling agents, where the compounds areused in a process for detection of nucleic acid or as a label which isprepared for labeling a compound to provide a fluorescent signal. Byhaving multiple markers capable of simultaneous detection, manyanalytical applications are attainable. For example, such applicationsinclude fluorescence immunoassay, fluorescence in situ hybridization,and flow cytometric analysis of cell populations, to list a few.

By appropriate combinations of the subject compositions, one can employdyes which share a common excitation wavelength, exploiting energytransfer to achieve readily resolvable emission wavelengths. Inaddition, with the dsDNA-dye complex, excitation of the donor leads togreatly enhanced emission from the acceptor, with the donor fluorescencesubstantially quenched as compared to the appropriate monomer dye in thesame environment. Thus, by using combinations of the subject dyes,high-sensitivity multiplex detection of different dsDNA fragments can beachieved.

The subject compositions can find use in separations employing anelectrical field, e.g. electrophoresis. In employing the subjectcompounds, the nucleic acid, usually DNA, and the dye may be broughttogether in appropriately buffered medium and incubated for sufficienttime for the dye to non-covalently bind and intercalate in the nucleicacid. The ratio of dye to double stranded nucleic acid may be variedwidely ranging from about one molecule of dye per base pair to as littleas one molecule of dye per 400 base pairs, or fewer, usually as few asone molecule of dye per 100 base pairs depending upon the desired degreeof sensitivity. Below about 15 bp/dye molecule, the increase in emissionupon further addition of dye is not as efficient as above 15 bp/dye. Dyepresent in excess of one dye for four base pairs or more, may result intotal quenching, so that any increase in the amount of dye above a molarratio of one dye molecule for four base pairs may not be desirable.However, the amount of dye combined with the DNA may be in a ratio of 1per 2 base pairs or even 1 per 1 base pair or even greater ratios, wherequenching is not observed. Generally, the amount of dye will range fromabout one molecule for 4 to 100 base pairs, usually about 10 to 50 basepairs, for optimum results.

One may combine different samples with different dyes, followed bycombining the different samples to be electrically separated. Thus, inthe same channel, where an electrophoresis is carried out, one candetect the various bands with light of the same wavelength used forirradiation, by detecting the differences in fluorescent wavelength fromthe various bands.

The amount of nucleic acid will generally be conventional amountsemployed for electrophoresis, generally ranging from about 5 pg/μl to 5ng/μl. Because of the fluorescent efficiency, capillary electrophoresiscan be performed efficiently. Various conventional buffers may beemployed, such as tris-acetate or tris-borate, generally present in therange of about 1-50 mM, more usually in the range of about 1-20 mM, toprovide a pH in the range of about 5-10, more usually about 7-9. Also, ametal ion chelator may be present in a minor amount, generally fromabout 0.05-0.5 mM. Conveniently, EDTA may be employed.

The dye and nucleic acid may be incubated, usually for at least about 5minutes and not more than about 2 hours, where complex formation willnormally be complete in less than about 1 hour, usually in about 30min., at room temperature. The incubated solution may be used directlyor further diluted, as appropriate, prior to application to the gel.

The electrophoresis may be performed in any convenient and conventionalmanner, where the bands may now be detected by fluorescence of thenon-covalently bound and intercalated dye. The electrophoresis ensuresthat unbound dye is removed from the region of the bands and the dye isfound to be retained in the nucleic acid, so that individual bands mayreadily be detected by fluorescence scanning.

Instead of incubating the nucleic acid with the dye prior to applyingthe nucleic acid to the gel, one may apply the dye after having carriedout the separation. Since the intercalated dye will have a substantiallydifferent absorption-emission range (and much enhanced fluorescenceintensity) from the unintercalated dye, one can readily detect theintercalated dye, even in the presence of significant amounts of thenon-intercalated dye.

Any conventional detection system may be employed for detecting theindividual bands. Depending on the particular dye employed, theexcitation light will be chosen to be within a major absorption band ofthe absorbing dye.

Of particular interest is the use of a confocal laser scanningfluorescence imaging system. A system which has been found to beconvenient employs a long-pass dichroic beam splitter to reflect thelaser beam down through a microscope objective and onto the sample. Thefluorescence emission is collected by the objective and passed throughthe beam splitter to a photodetector. The fluorescence emission is thenpassed through a spatial filter to effect confocal detection in along-pass or band-pass color or interference filter before reaching aphotomultiplier tube. An appropriate servomotor-driven XY translationstage is employed with a 2.5 μm resolution to translate the gel past thelaser beam at a convenient speed, nearly about 1-5 cm/sec. Amicrocomputer may be employed to control the XY translation stage and toacquire and display images. The fluorescence images may then bepseudo-colored and coded to represent different intensity levels andcontrast stretched with a histogram equalization method to enhance theimages. To quantitate the image data, the image columns that enclose thenucleic acid bands may be extracted and integrated.

The nucleic acid may be readily isolated free of the intercalatedfluorescent dye for further use. One may use the Geneclean® kit forrecovery of 50% or better of the nucleic acid. By combining theintercalated dye containing nucleic acid with Glassmilk in an aqueoussolution of alkali metal iodide, e.g. 1-10 ng nucleic acid (1-5 μg/mlnucleic acid) and about 1-10 μg/ml of Glassmilk, incubating withagitation for about 5-60 mins. followed by centrifugation, the resultingpellet is isolated. After resuspending the pellet in an appropriateethanolic buffered aqueous solution (e.g. 1:1) followed bycentrifugation and repeating this washing procedure, the nucleic acid isobtained substantially free of the fluorescent dye.

By virtue of the use of the subject intercalating fluorescent dyes inthe electrophoresis, greatly enhanced sensitivities are achieved due tothe much higher level of fluorescence intensity which is obtained. Sizesand amounts of DNA fragments in mixtures of unknown composition can bedetermined with a total amount of material ranging from 100 pg to 1 ngdepending on the complexity of the mixture and the size range of thefragments. Thus, the subject method can find application in thedetection of nucleic acid of less than about 5 ng, particularly lessthan about 1 ng, frequently less than about 100 pg, even less than about50 pg.

Instead of employing the subject dyes for detection of nucleic acidbands in electrophoresis, compositions comprising dsDNA and the subjectdyes at substantial saturation may be employed, where the dsDNA isjoined to an entity for binding to another entity, either covalently ornon-covalently. The entities will be either referred to as specificbinding pairs, since the entities will have specific affinity for acomplementary entity, as compared to diverse other types of molecules,or covalently binding functionalities for reacting with other molecules,such as polypeptides or saccharides.

The specific binding pairs may involve a wide variety of molecules,which are arbitrarily called ligands and receptors. For the subjectinvention, the ligands and receptors may include a wide variety ofproteins, such as antibodies, specific binding proteins, such as surfacemembrane protein receptors, lectins, blood proteins, and the like,carbohydrates, small organic molecules, both naturally occurring andsynthetic to which proteins specifically bind, either naturallyoccurring protein receptors or antibodies, nucleic acids which mayhybridize or specifically bind to an homologous or partially homologoussequence usually having at least about 30% complementarity, preferablyat least about 50% complementarity over the complementary region, andthe like. In effect, any two molecules which have a specific bindingaffinity may be employed, so that the label may be used for detection ofthe presence of the complementary member. The desired specificity may bevaried widely, depending upon the particular nature of the molecules tobe detected, the information desired about the nature of the sample, orthe like.

The labels may be used for detecting any of a wide variety of moleculesin a wide variety of samples, which includes physiological samples, e.g.blood, plasma, urine, spinal fluid, saliva, feces, mucus, etc., wastesamples, from processing, garbage, soil, water, etc., contaminants inproducts, such as food, drugs, etc.

Depending upon the fluorescence intensity one desires, one can vary thelength of the dsDNA and the level of non-covalent binding andintercalation to increase the fluorescence intensity per molecule.Usually, there will be at least about 16 base pairs, more usually atleast 20 base pairs, and one may have dsDNA of at least about 1 kbp oreven 2 kbp or more. The particular length of the dsDNA is not criticalto this invention and may be varied in accordance with the fluorescenceintensity desired per molecule, purpose of the label, convenience, andthe like. It is found that with some dyes, e.g. ethidium-acridineheterodimer, there is an increase in fluorescence intensity by havingA-T pairs. Thus, one may provide for a poly A-T.poly A-T dimer to beused as the label. However, if one wishes to further increase thestability of the dsDNA, beyond that which the intercalating dimerprovides, one can use a combination of AT and GC pairs or a polyG-C.poly G-C dsDNA. Alternatively, one may use any source of random DNA,such as calf thymus DNA, E. coli DNA, etc.

The dsDNA should provide for means for binding to another molecule. Thiscan be achieved in a wide variety of ways, depending upon the manner inwhich the label is to be employed. For example, the dsDNA may includebiotin conjugated nucleotides, one or more biotins, where the biotinwill bind to avidin or streptavidin (hereafter both will be referred toas "avidin"). The biotins may vary from one biotin per nucleotide to0.1% of the nucleotides depending on the nature of the procedures,conditions, etc. Alteratively, any molecule may be employed,particularly a small organic molecule (less than about 2 kdal) which isunlikely to be encountered in the sample of interest, where the smallorganic molecule has a specific receptor or antibody, particularlymonoclonal antibody, to which it specifically binds. Thus, thyroxine,corticosteroids, estrogens, retinoic acid, mannose and the like may beused with proteins which bind specifically to such molecules.Alternatively, synthetic molecules may be employed for which antibodieshave been produced, such as 2,4-dinitrophenyl, barbiturate,phosphatidylcholine, etc. These molecules may be included duringsynthesis of the DNA by being linked to an internal or terminalnucleotide, where the DNA is synthesized in accordance with conventionalautomatic procedures, or may be added after synthesis of the DNA bylinking to either available hydroxyl or amino groups.

The binding entity may be an active functionality for covalently bondingto a molecule having at functionality capable of forming a stablecovalent link, such as amino, hydroxyl, thio, carboxyl, activated olefinor aryl, or the like where the functionality is other than a naturallyoccurring functionality of the nucleotide. The label may be modifiedwith an activated olefin, such as maleyl, for reaction with a thiolgroup, a carboxyl for reaction with an amine, or the like. In thismanner, many different types of molecules may be fluorescently labeledfor use in diagnostics, both competitive assays and non-competitiveassays, histology, cytology, separations e.g. electrophoresis, HPLC,FACS, and the like.

The strands of DNA may take various structures. In many situations, thedsDNA may comprise two strands, where the strands may be completely oronly partially overlapping, where the ends may extend in the 5' and/or3' directions, so that one strand may be substantially longer than theother strand, where the other strand may bind either 5' proximal, 3'proximal or centrally. Alternatively, the two strands may overlap toprovide for staggered ends, where the single stranded portions of theDNA may then be used to bind to complementary sequences. Alternatively,one may provide a single strand with an inverted repeat, so that thestrand loops back on itself to provide the double stranded portion. Thehairpin structure may be used solely for labeling, or a single strandedportion of the hairpin may be employed for hybridizing to acomplementary sequence. The hybridizing single stranded portion may bean extension at either the 5' or 3' end to provide for a staggeredterminus or may be present in the loop of the hairpin.

The subject labels may be used in a wide variety of environments andcontexts to provide for high levels of fluorescence intensity withoutinterference from the molecules to which the labels bind, eitherdirectly or indirectly, the media employed, the conditions employed, andthe like. Thus, the subject labels may be employed in specific bindingpair assays, where the label may be readily linked to another moleculethrough a specific binding pair combination. For example, in diagnosticassays, one may combine an avidin conjugated antibody, where theantibody binds to a molecule of interest, to a biotin labeled DNA dyecomposition to provide for fluorescent labeled antibody.

Alternatively, the antibody may be labeled with biotin, so that avidinmay act as a bridge between the biotin labeled antibody and the biotinlabeled DNA dye composition. In this way, the fluorescent label may beadded after combining the sample with a complementary specific bindingpair member and carrying out the assay, followed by addition of labeland removal of any nonspecifically bound label.

Where a single stranded DNA sequence is provided as part of the label,this can be used for hybridizing to complementary DNA or RNA sequences.The presence of the non-covalently bound and intercalated dye greatlyenhances the stability of the dsDNA. Thus, one can introduce the subjectlabels into a denaturation medium under conditions where thenon-covalently bound and intercalated dsDNA will be stable, while thesample DNA may be denatured to provide for single strands. Where singlestranded DNA or RNA is present, there will be no need for provided fordenaturation conditions. Therefore, the subject molecules may be used asprobes to identify DNA sequences under a wide variety of conditions,including electrophoresis, polymerase chain reactions, where the singlestranded sequence may serve as a primer, in Southern blotting, Northernblotting and the like.

Instead of having non-covalent complexes between the non-nucleic acidspecific binding pair member and the DNA dye aggregate, one can providefor covalent bonding. Thus, by providing for activated groups such ascarboxy, diazo, azido, activated ethylene, or the like, the fluorescentmoiety may be readily linked to other molecules, such as proteins,sugars, lipids, or the like by employing conventional linking groupsresulting in amides, amines, diazo, esters, thioethers, or insertioninto a carbon-hydrogen bond or addition to a double bond, and the like.For example, one may introduce a thiol group at either the 3' or 5'terminus of a synthetic oligonucleotide, synthesize the complementarystrand and form a non-covalently bound and intercalated dye complex. Thethiol group on the DNA can then be reacted with a maleimide modifiedprotein, e.g. an antibody. One can add an acylmethylazide and uponphototypes produce a nitrene which would randomly insert or add to adouble bond. Other techniques may follow conventional procedures foundin the literature.

The subject DNA dye composition may also be used in situations where onewishes to transfer energy or receive energy from another molecule. Thus,the subject compositions may be used with other fluorescent dyesubstituted molecules, e.g. dye intercalated DNA molecules, for receiptor transfer of excitation energy, or with other fluorescent molecules,so as to extend the shift between the excitation light and the emissionlight. This technique may be used in diagnostic assays, or where onewishes to determine the spatial relationship between two entities, e.g.epitopes, surface membrane receptors, etc.

One may also use the subject labels in a fluorescence activated cellsorter to provide for greatly enhanced sensitivity as a result of thesubstantially increased fluorescence intensity. Again, one may useligands for surface membrane receptor proteins, sugars for lectins,antibodies for epitopes present on the surface of the cell, or the like,where the subject labels may be bound covalently or non-covalently, tothe molecule which binds to the cell component.

With the subject compositions one can also detect proteins totranscriptional initiation elements, e.g. promoters, operators,enhancers, etc. By having labeled dsDNA, according to the subjectinvention, mixed with labeled proteins, labeled with a fluorescentmolecule emitting at a different wavelength from the non-covalentlybound and intercalated fluorescer, or other appropriate label, one candetermine the presence of transcription factors and cofactors. Forexample, one can gel electrophorese the mixture and identify thepresence of the protein bound to DNA by virtue of the double labellingand band shift.

One may also use the subject fluorescent non-covalently bound andintercalated DNA for in situ hybridization studies, intermoleculartransfer of fluorescent molecules from one doubly stranded nucleic acidmolecule to another, e.g. for transferring fluorescent dye without thefluorescer being transferred to the medium. This may find use in makingchromosomes with triplex formation, in transferring to nuclei acid in agel or on a membrane, etc. The fluorescer intercalated DNA may be boundto a particle, e.g. magnetic, to be removed after use as a transferagent.

The subject compounds may be used with advantage with a confocalfluorescence imaging system, where less than 100 pg of DNA can bedetected with some dyes while with other combinations, less than about 5pg of DNA can be detected. In histology and cytology, the subjectfluorescent labels provide for high-sensitivity in detecting targetepitopes, particularly at low levels.

Besides using the dyes individually, the dyes may be used in combinationfor a wide variety of applications, where one wishes to obtain at leasttwo bits of information concerning the sample or composition ofinterest. Since the intercalated dyes have different absorption andemission spectra from the non-intercalated dyes, one can detect thepresence of the two dimeric compounds when intercalated into DNA, whilein the absence of DNA, there would be substantial overlap, so that onlypoor discrimination would be obtained. Furthermore, one need not use theheterodimers of the subject invention solely, since the heterodimers maybe used with homodimers, where the heterodimer and homodimer have thesame absorbing fluorophore or different absorbing fluorophore excited bya common wavelength range, but obviously differing in the emittingfluorophore. Thus, ethidium dimer, thiazole orange dimer, thiazole bluedimer, oxazole yellow dimer, and the like may be used in conjunctionwith heterodimers, where usually the same absorbing fluorophore ispresent, although in many instances, a different absorbing fluorophorewill suffice, where the two absorbing fluorophores have overlappingabsorption peaks.

By using the combinations employing the subject dyes, detection of twodifferent events may be obtained in a number of different environments.Of particular interest is flow cytometry, where a single exciting lightmay be used and the fluorescence determined as to two or more events.The events may involve binding events to two different epitopes of thesame or different protein, where a single protein may be involved or anaggregation of proteins, as is present in viruses and cells orsignificant fragments thereof. In this way, one may select for particlessuch as cells, by virtue of the presence of two different markers, usinga single exciting laser. Similarly, in histology and cytology, one maydetermine the presence of different proteins which may be present on thesame or different cells or present extracellularly. As appropriate, onemay inject the dyes bound to a specific binding molecule into alaboratory animal to follow the migration of different molecules orcells, where one is interested in the presence of the two differenttargets at a particular site. Numerous other applications, where singleexcitation light is desired, while a plurality of different informationvalues is desired concurrently, have been generally described inliterature and will be further developed as the subject inventionbecomes available.

For many applications, a plurality of fluorescent molecules will bedesirable. Kits can be provided where the fluorescent molecules in thekit are characterized by having absorption maxima within about 25 nm, sothat excitation light of a relatively narrow bandwidth may be used,generally of not more than about 30 nm, usually of not more than about20 nm. The absorbing fluorophore may be the same or different.

The kit will have two or more fluorescent multimers, each having fromtwo to four, usually two to three, fluorophores, wherein at least one ofthe fluorophores is able to bind, usually intercalate, into dsDNA. Atleast one of the multimers will be a heteromultimer being characterizedby having a Stokes shift of at least about 25 nm, capable of bindingdsDNA at a ratio of at least one dye per 200 bp dsDNA withoutdiminishing the fluorescence as compared to one fluorophore per dsDNAmolecule on a per dye basis, and the fluorophores absorb and emit atdifferent wavelengths, when bound or unbound to dsDNA. Besides the atleast one heteromultimer, the other multimers may be homomultimers, ofparticular interest are homo- and heterodimers and heterotrimers.

The following examples are offered by way illustration and not by waylimitation.

EXPERIMENTAL

SYNTHESIS OF DYES

Materials and Methods. The starting materials including3-methyl-benzothiazole-2-thione, lepidine,3,8-diamino-9-phenylphenanthridine, fluorescein isothiocyanate isomer I,diethylenetriamine, tetramethyl-1,3-diaminopropane, 1,3-diaminopropane,anhydrous HBr/acetic acid were purchased from Aldrich and used withoutfurther purification. Anhydrous methanol, triethylamine, and pyridinewere distilled from sodium and stored under nitrogen. Anhydrousnitrobenzene was freshly distilled from P₂ O₅. All anhydrous reactionswere run in oven dried glassware under a nitrogen atmosphere. Reactionswere monitored by TLC (Merck A₂₅₄) under short and long wavelength UVlight. Flash chromatography was performed on 220-440 mesh silica gel 60from Fluka. Intermediate products which gave single spots on TLC wereidentified by their ¹ H-NMR spectra measured with an AMX-300 instrument.UV/VIS absorption spectra were measured with a Perkin Elmer Lambda 6spectrophotometer and fluorescence emission spectra with a Perkin ElmerMFP 44B spectrofluorometer.

Synthesis of Heterodimers. As outlined in FIG. 1, the N-3-iodopropylderivatized lepidine (1), reacted in under 15 minutes with the N-methyl2-methylthio benzothiazole (2) or N-methyl 2-(N'-phenyl, N'-acetyl,3-azopropylidene benzothiazole (4) to yield the iodopropyl thiazoleorange intermediate (3), or iodopropyl thiazole blue intermediate (5)(TB), in good yields following the method of Brooker et al. ((1942) JACS64:199-210; (1941) JACS 63:3192-3203). Compound (1) was produced in highyield by alkylation of lepidine with 5 equivalents of 1,3-diiodopropanein ref luxing dioxane, while compound (2) was formed quantitatively when3 equivalents of iodomethane were reacted with3-methylbenzothiazole-2-thione in refluxing 200 proof ethanol andprecipitated with ether. Compound (2) was also used as an intermediatein the synthesis of (3) (Brooker et al., supra).

FIG. 1. Synthesis of thiazole monomer intermediates.

a) Suspend in absolute EtOH, add 1 equivalent TEA, stir at roomtemperature for 15 minutes, precipitate with ether, recrystallize fromacetone/ether; yield 80% 3, 60% 5.

To synthesize the thiazole orange-thiazole blue heterodimer, theiodopropropyl thiazole orange derivative (4) was reacted with excesstetramethyl-1,3-diaminopropane (Scheme 2) to produce the intermediatetetramethyldiaminopropyl thiazole orange derivative (6). Compound (6)(TO6), after purification by recrystallization, reacted with thethiazole blue derivative (5) to produce a good yield of the thiazoleorange linked thiazole blue heterodimer TOTAB (7). Dimerization of (4)(as described in Rye et al., Nucleic Acids Res. (1992) 20:2803-2812) wasused to synthesize nearly quantitatively the homodimer TOTO (8)employing 0.5 equivalents of tetramethyl-1,3-diaminopropane.

FIG. 2. Synthesis of the thiazole dimers. a) Suspend in anhydrous MeOH,add 6 equivalents tetramethyl-1,3-propanediamine, reflux 6 hours,precipitate acetone/ether, recrystallize MeOH:CH₂ Cl₂ (1:10) /acetone;yield 80% 6. b) Suspend in anhydrous MeOH, add 1 equivalent (5), reflux10 hours, precipitate with ether, triturate solid with MeOH:CH₂ Cl₂(1:10), flash column EtOAc:AcOH:H₂ O (1:2:2); yield 70% 7. c) Suspend inanhydrous MeOH, add 0.5 equivalents tetramethyl-1,3-propanediamine,reflux 18 hours, precipitate with acetone, titurate solid with MeOH:CH₂Cl₂ (1:10), recrystallize from MeOH/acetone; yield 95% 8.

The thiazole orange-ethidium heterodimer was obtained as outlined inFIG. 3 from 3,8-diamino-9-phenylphenanthridine (9) via intermediate(10). In general, the synthesis of the thiazole orange-ethidiumheterodimer (11) followed the method of Gaugai et al. ((1978)Biochemistry 17:5071-5078) except that carbobenzyloxy groups were usedto protect the amino substituents in place of acetate or carboethoxygroups. After coupling (10) with the thiazole derivative (3), the crudesolid dicarbobenzyloxy-protected intermediate was deprotected (anhydrousHBr/AcOH) to produce a low unoptimized yield of the desired thiazoleorange-ethidium heterodimer TOED (11) and substantial amounts ofundesired byproducts. Employing tetramethyldiaminopropane in place ofdiaminopropane as a linker in the synthesis of thiazole orange-ethidiumheterodimer gave an improved yield of the analogue, TOED-2, with aquaternized methylenediamine linker. The fluorescein-ethidiumheterodimer was synthesized as described in FIG. 4.

FIG. 3. Synthesis of the thiazole-ethidium heterodimer. a) Suspend inanhydrous pyridine, add dropwise 2.2 equivalents carbobenzyloxychlorideat 0° C., stir to room temperature over 12 hours, precipitate withether/pet ether, suspend solid in CH₂ Cl₂ and wash with 10% NaHCO₃, dryorganic layer, concentrate, flash column MeOH:CH₂ Cl₂ (1:50). b) Suspendin anhydrous nitrobenzene, add 5 equivalents 1,3-diiodopropane, heat at160° C. for 4 hours, precipitate with ether, flash column MeOH:CH₂ Cl₂(1:10). c) Suspend in anhydrous MeOH, add 10 equivalents1,3-diaminopropane, reflux 7 hours, precipitate H₂ O, suspend EtOH andacidify with concentrated HCl, precipitate with ether, flash columnEtOAc:AcOH:H₂ O (6:3:2); yield 22% (10) from (9). d) Suspend in MeOH,add 3 equivalents 1N NaOH, precipitate/wash solid with H₂ O, dry solidat 60° C. in oven. e) Suspend in anhydrous MeOH, add 0.7 equivalents 3,reflux 10 hours, precipitate ether/pet ether; used without furtherpurification. f) Suspend in anhydrous HBr/AcOH, stir at room temperaturefor 1 hour, concentrate, suspend in CH₂ Cl₂ precipitate with TEA/ether,flash column EtOAc:AcOH:H₂ O (6:3:2).

FIG. 4. Synthesis of the fluorescein-ethidium heterodimer. Steps (a) and(b) are performed exactly as described for steps a and b in Scheme 3. c)Suspend in anhydrous MeOH, add 10 equivalents of diethylenetriamine,reflux 7 hours, precipitate H₂ O, suspend in EtOH and acidify withconcentrated HCl, precipitate with ether, flash column EtOAc:AcOH:H₂ O(6:3:2); yield 28% (12) from (9). d) Suspend in MeOH, add 4 equivalents1N NaOH, precipitate/wash solid with H₂ O, dry solid at 60° C. in oven.e) Suspend in anhydrous MeOH, add 1.1 equivalents of FITC, stir at roomtemperature 2 hours, acidify with concentrated HCl, precipitate withether, solid used without further purification. f) Suspend in anhydrousHBr/AcOH, stir at room temperature for 1 hour, concentrate, suspend inCH₂ Cl₂, precipitate with TEA/ether, flash column EtOAc:AcOH:H₂ O(7:3:2).

The linked structure of the dyes, TOTAB (7), TOED (11) and FED (13), wasestablished by the presence of both monomer UV/visible absorptions inthe chromatographically pure dimers which were shown to contain none ofthe monomeric starting materials thiazole orange (6), thiazole blue (5),ethidium derivatives (10) or (12), or fluorescein isothiocyanate by TLC(in 1:1 v/v MeOH:CH₂ Cl₂ and EtOAc:AcOH:H₂ O 1:2:2, by volume). Thevisible absorption maxima in MeOH at 507 nm and 633 nm exhibited byTOTAB, and the 294 nm and 486 nm absorptions of FED, clearly indicatedthe presence of linked chromophores in the dyes (See Table 1).

                  TABLE 1                                                         ______________________________________                                        Solvent                                                                       MeOH            TAE Buffer   TAE Buffer/DNA                                   Dye    λ max                                                                          ε                                                                              λ max                                                                         ε                                                                           λ max                                                                        ε                          ______________________________________                                        TOTAB  302     22 000   302    15 000                                         7      505     77 600   506    57 100                                                                              514   60 800                                    633     101 000  644    55 800                                                                              646   46 600                             TOED   295     62 000   288    53 600                                         11     476     50 500   472    54 100                                                                              472   sh                                        507     56 400   507    sh    515   57 500                             TB     304     12 800                                                         5      633     160 500  304    10 100                                         (505, ε = 2 600)                                                                      628      75 300  639   70 200                                 TO     290     13 200   288    10 500                                         6      507     76 000   507    64 300                                                                              515   61 700                             FED    294     72 000   288    68 800                                         13     486     13 200   495    72 300                                                                              492   48 600                             FITC   280     23 200   280    24 200                                                456      2 800   492    72 000                                                                              492   72 000                             Ethidium                                                                             294     48 800   284    44 600                                         Bromide                                                                              520      5 500   475     5 500                                                                              516    4 200                             ______________________________________                                    

Absorption spectra of equal concentrations of the monomers were added toclosely approximate the spectra of an equal concentration of the linkeddimers. Comparison of the absorption spectra of thiazole orange (6) andthiazole blue monomer (5) established that less than 3% of theabsorption at 505 nm of the TOTAB dimer was due to the thiazole blueportion of the dimer. Thus, the extinction coefficient of the thiazoleorange monomer (6) plus the extinction coefficient of the thiazole bluemonomer (5) at 505 nm was assigned to the 505 nm absorption of the TOTABdimer (ε=77 600 M⁻¹ cm⁻¹) and used in all subsequent TOTAB concentrationdeterminations. For the TOED dimer, addition of the separate spectrataken of equal concentrations of ethidium bromide and (6) closelyapproximated that of an equal concentration of the dimer in the UVregion of the spectrum, and was used to determine an extinctioncoefficient (ε=62 000 M⁻¹ cm⁻¹) for absorption at 295 nm for the TOEDdimer. Calculation of the extinction coefficients at the UV and visibleabsorption maxima of FED (13) were complicated by the high sensitivityof the visible absorption spectrum of the fluorescein chromophore tosolvent. The ultraviolet absorption spectrum of FED in methanol and inTAE was closely approximated by the sum of the absorption spectra ofequal concentrations of fluorescein isothiocyanate and ethidium bromide.A pronounced tendency of the dyes to retain salts produced during thevarious stages of the syntheses precluded easy direct measurement oftheir extinction coefficients based on dry weight.

SPECTRA OF dsDNA-HETERODIMER DYE COMPLEXES

Fluorescence spectra of TOTAB, TOED and FED bound to calf thymus dsDNAat 100 bp:dye, with excitation at 488 nm, were determined. Fluorescencespectra of the free dimers were uninformative as the emissionsoverlapped to produce a broad fluorescence centered at 660 nm for TOTABand 630 nm for TOED. The large bathochromic shift for the thiazoleorange portion of each dimer upon, binding to DNA produced two emissionmaxima in the spectra of the bound dyes. Under these conditions, TOTABfluoresced maximally at 532 nm and 662 nm corresponding to thiazoleorange and thiazole blue emissions. TOED fluoresced maximally at 532 nmand 610 nm corresponding to thiazole orange and ethidium emissions.Tight binding of the dyes to DNA was indicated by the 384 fold increasein the long wavelength fluorescence emission of TOTAB, and the 227 foldincrease in the long wavelength fluorescence emission of TOED uponbinding DNA relative to the fluorescence of the dyes free in solution.The strong fluorescence enhancement of bound versus free TOTAB and TOEDdyes indicated tight binding of both chromophores to the DNA. The lackof a blue shift in the thiazole blue portion of TOTAB upon binding DNA(emission at 660 nm free versus 662 nm bound) suggested that thisportion of the molecule may in fact be bound in a non-intercalative modein the minor grove of the DNA exposed at least in part to the externalaqueous environment.

From comparison of the absorption spectra of bound dyes to thefluorescence emission of the bound dyes upon irradiation at 488 nm itwas evident that energy transfer occurred between donor and acceptorchromophores in both dyes. The observed enhancement of the thiazole blueportion of the TOTAB emission at 662 nm, relative to the 532 nm emissionproduced by 488 nm excitation of the thiazole orange portion of the dye,was the clear result of energy transfer given the absorption profile ofthe bound dye. Similarly, the visible absorption profile of TOED boundto DNA was dominated by the thiazole orange absorption at 488 nm (εdonor:ε acceptor=14:1), yet energy transfer upon excitation at 488 nmresulted in a larger ethidium emission at 610 nm then the thiazoleorange emission at 532 nm. The observation of highly efficient energytransfer at a DNA bp:dye ratio of 100:1, provides independent evidencethat the donor and acceptor chromophores in TOTAB and TOED are linked toeach other. Plots of the emission spectra of the unlinked thiazoleorange monomer T06 (compound 6) bound to DNA when compared at the sameconcentration of TOTAB and TOED bound to DNA demonstrated that thethiazole orange emission of the bound heterodimers was more than 90%quenched at well below saturation of binding sites (100 bp:dye) on theDNA Similarly, a comparison of the emission spectra of FED and the sameconcentration of fluorescein isothiocyanate demonstrates that greaterthan 90% quenching of the fluorescein emission occurrs in the bounddimer. A comparison of the fluorescence emissions of the heterodimers toethidium dimer bound to DNA was also examined: the TOTAB dimerfluoresced over 9 time as brightly as ethidium dimer in solution at itsemission maximum while the TOED dimer fluoresced 8 times as brightly.Based on the ratio of 610 nm emission of ethidium fluorescence in TOEDversus the 610 nm emission of the two ethidium chromophores in theethidium dimer (taking into account the extinction coefficient ratio ofethidium to ethidium dimer; 5500:8900), a 15-fold fluorescenceenhancement of the ethidium chromophore emission in the TOED dimer,bound to DNA at 100 bp:dye, was calculated. This result exemplifiesconvincingly the ability of energy transfer to increase fluorescenceyield of a weakly absorbing chromophore.

Titrations of calf thymus dsDNA with TOTAB and TOED, monitored at theacceptor emission maxima or plotted as the ratio of acceptor/donoremission maxima, let to a variety of significant observations. As siteson the DNA become saturated with dye, the emissions of both donor andacceptor chromophore per mole of added dye become increasingly quenchedparticularly the donor emissions. Titrations of calf thymus DNAmonitored by the acceptor chromophore emissions and normalized per moleof added dye indicate an approximately linear relationship offluorescence per mole of added dye from 100 to 20 bp per dye, indicatingthat in solution the binding of the dyes does not show strong sequencespecificity. Titration was performed by adding concentrated aliquots ofstock dilutions of the dye to calf thymus DNA (c=5×10⁻⁶ M bp) in 0.5 mLof 4 mM TAE and incubating the mixture for 15 minutes prior to recordingeach spectrum. The two sets of titration data obtained were normalizedrelative to each other by recording the emission spectra of the two dyesbound to calf thymus DNA at 100 bp dye under identical conditions. Assites on the DNA become saturated beyond 20 bp:dye, the amount ofemission per mole of added dye diminishes rapidly. Secondary sitebinding is a common property of intercalator dyes as primary sitesbecome saturated with dye (Gaugain et al. (1978) Biochemistry17:5078-5088). Judging from the rapid drop in emission as primaryintercalative sites on the DNA are saturated, it appears that for bothdyes, binding to secondary non-fluorescent sites caused externalquenching of the primary site bound dye.

Analysis of the ratio of emissions of the donor and acceptorchromophores as a function of the ratio of bp DNA to dye (see above forconditions) showed that the amount of quenching of the thiazole orangedonor emission of the dyes compared to the quenching of acceptoremission is strongly dependent on the fractional saturation of bindingsites on the DNA. As the percentage of neighboring acceptor chromophoresincreases, the thiazole orange donor emission becomes increasinglyquenched. At saturating levels of dye, the thiazole orange emission at532 is nearly quenched for both TOTAB and TOED bound to DNA. Thus, forthe titration employing TOTAB at 3 bp:dye, a maximal ratio ofacceptor:donor fluorescence of 17:1 is reached, while in the titrationwith TOED a maximal ratio of acceptor:donor fluorescence of 39.5:1 isattained. For both TOTAB and TOED dyes bound to DNA, in the presence ofexcess dye (at 0.5 bp DNA:dye) emissions of both donor and acceptor arealmost completely quenched.

The spectrum of the fluorescein-ethidium heterodimer (FED, compound 13)was determined. Comparison on an equimolar basis of the fluorescenceemission spectra of FED versus that of ethidium homodimer, both inpresence of calf thymus DNA at 100 bp DNA:dye, showed that above 620 nmFED emits over 7 times more fluorescence than ethidium homodimer.

DETECTION OF dsDNA ON GELS

Materials and Methods. Electrophoretic separations and detection ofdsDNA-dye complexes are performed by following the procedures describedby Rye et al., supra. Electrophoresis is performed in a Mini-Protean IIapparatus (BioRad, Richmond, Calif.) in 1 mm thick, vertical 0.9%agarose gels run at 10 V/cm in 40 mM Tris-acetate-EDTA (TAE), pH 8.2.Gels were pre-electrophoresed for 1 hour prior to sample loading.Working stocks of dyes (c=1×10⁻⁷ M) are freshly prepared fromconcentrated stock dyes (c=1×10⁻⁴ M in MeOH or DMSO), before eachexperiment by dilution into 4 mM TAE, pH 8.2. The known instability ofthe cyanine and ethidium dyes in basic buffers (Rye et al., supra)mandates that working stocks in TAE be freshly prepared. For a typical 1ng λ DNA/HindIII DNA load, concentrated DNA (2 μl of 10 ng/μl DNA) isadded to the dye in 4 mM TAE pH 8.2. The dye is diluted from the workingstock to give the correct DNA bp:dye ratio upon addition of the DNA in afinal volume of 75 μl of a 0.27 ng/μl DNA (4.19×10⁻⁷ M bp) plus dye(4.19×10⁻⁸ M dye for 10 bp:dye) mixture which is subsequently incubatedin the dark for 30 minutes. After the 30 minute incubation period, 25 μlof 15% aqueous Ficoll solution is added to give 100 μl of a 0.2 ng/μlDNA-dye mixture containing 5% Ficoll. A 5 μl (1 ng DNA load) sample ofthis mixture is loaded on the agarose gel and electrophoresed in thedark for 1 hr. The gel is subsequently scanned with a confocal imagingsystem (Rye et al., supra). The data are analyzed as previouslydescribed in Rye et al., supra.

Results. Initial electrophoresis experiments with a λ DNA/HindIII laddershowed that the heterodimeric dye-DNA complexes, in particular, thecyanine dimers TOTO and TOTAB dye-DNA complexes, gave variable resultsdue to a tendency of the complexes to streak and precipitate in thewells of the gel. To produce consistent results, for TOTAB, addition of100 mM NaCl to the dye solution in 4 mM TAE prior to incubation with DNAresulted in complete elimination of the streaking and precipitationproblems. The use of 40 mM TAE incubation buffer had been previouslyobserved to improve band patterns in studies with oxazole yellow dimer(Rye et al., supra). Further studies showed that band sharpness andsignal intensity were not improved at <50 mM NaCl, but were optimal at80 to 100 mM salt. Signal intensity of the bands dropped at >150 mM NaClin 4 mM TAE or 40 mM TAE in the incubation mixture, likely due to alower binding constant of the dye for DNA at the higher saltconcentrations. For all dyes studied, addition of 50-100 mM NaCl to 4 mMTAE buffer in the incubation mixture produced consistent results and wassubsequently employed in all experiments.

A comparison of the signal intensities of dsDNA complexes of TOTAB, TOEDand ethidium dimer bound to the same amount of λ DNA/HindIII DNA all ata 5 bp:dye ratio and run on the same gel, showed that TOTAB was 4-5times as bright as ethidium dimer, but TOED was found to be onlyslightly brighter than ethidium dimer. The modest increase insensitivity of DNA detection with TOED was due to the rapid loss of TOEDfrom the DNA during electrophoresis. In contrast, TOED-2 formed a muchmore stable complex with DNA and allowed two-fold higher sensitivity ofDNA detection than is possible with ethidium homodimes. For TOTAB, aplot of λ DNA/HindIII fragment size versus signal intensity showed adirect linear relationship.

The sensitivity of the dyes was assayed by running a number of loads ofλ DNA/HindIII fragments complexed to dye at a constant ratio. Fragmentsstained with TOTAB at 5 bp:dye could be visualized down to 12 pg perband. TOED was less sensitive, for DNA loads of less than 1 ng only the23 kb fragment was detectable when stained with TOED at 5 bp:dye. The 23kb λ DNA/HindIII fragment band stained with TOED at 2 bp:dye wasdetectable down to 380 pg. Studies of time-dependence of band intensityfor TOED-stained λ DNA/Hind III fragments indicated slow dissociation ofthe dye in the course of electrophoresis.

Gel electrophoresis of mixtures of varying amounts of λ DNA/Hind IIIfragments with a constant amount of the dye was performed with TOTAB andTOED. For TOTAB, the band fluorescence intensity, calculated per mole ofdye, was constant for DNA dye ratios of 100 to 20 bp:dye and saturatedat 4-5 bp:dye.

The dependence of fragment mobility on DNA:dye ratio was explored forTOTAB. Standardized to a mobility of 1 for the fragments loaded with dyeat 100 bp:dye, mobility at 50 bp:dye was 1, at 20 bp:dye 0.99, at 10bp:dye 0.97, and at 5 bp:dye 0.92.

Complexes of FED with dsDNA were found to be more stable toelectrophoresis than complexes of dsDNA with ethidium bromide.

MULTIPLEX DETECTION AND SIZING OF dsDNA FRAGMENTS

A two-color multiplex experiment employing a 1 kb dsDNA ladder complexedwith TOTAB run together with λ DNA/HindIII fragments complexed withthiazole orange dimer was performed. A least squares fit of themobilities of the 1 kb ladder bands complexed with TOTAB plotted againstthe inverse log of size of the fragments allowed determination of thesize of the λ DNA/HindIII fragments stained with TOTO with a precisionof 2% or better.

It is evident from the above results that the subject invention providesunique opportunities for detecting a wide variety of molecules innumerous different environments. Of particular interest, is the abilityto use a single light source of narrow wavelength bandwidth to excite aplurality of molecules, where each of the molecules can be detectedindependently. Furthermore, by intercalating the subject dyes with DNA,the fluorescence yield is greatly enhanced.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to those of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

What is claimed is:
 1. A kit for use in multiplex fluorescence analysis,said kit comprising: at least two fluorescent multimers, each multimerhaving at least two fluorophores, at least one of said multimerscharacterized by:a) comprising at least two different fluorophores,which fluorophores absorb and emit at different wave lengths when boundto dsDNA; b) having a Stokes shift of at least about 25 nm; c) capableof intercalating dsDNA at a ratio of at least one dye per 200 bp; d)having from 2 to 4 fluorophores;wherein said fluorescent multimers canbe excited within the same narrow light bandwidth range of not more thanabout 30 nm when bound to dsDNA, and have emission maxima at least 25 nmapart wherein at least one of said multimers has a fluorophorecomprising benzthiazole, benoxazole, acridine, phenanthridine, orxanthene.